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  Introduction The Hsc70s are among the most important families of molecular chaperones. Members of the Hsc70 familyparticipate in numerous functions including folding of newlysynthesized proteins, transport of proteins into mitochondriaand the endoplasmic reticulum, formation of proteincomplexes in combination with the molecular chaperoneHsp90 and disaggregation of protein complexes such asclathrin-coated vesicles (Craig, 1989; Hartl et al., 1992). Hsp70was initially discovered as one of the most prominent heatshock proteins exhibiting markedly increased expression whenthe cell is stressed by an increase in temperature or otherstresses that lead to misfolding of proteins (Lindquist andCraig, 1988; Lindquist, 1986). An important function of Hsp70is protecting cells from the deleterious effects of misfolded andaggregated proteins (Hartl et al., 1994) by preventing themisfolding of proteins required for critical cell activities,disaggregating misfolded proteins and presenting irreversiblydamaged proteins to proteasomes for degradation.One set of proteins targeted by Hsp70 during heat shock ispresent in the nucleoli of mammalian cells. Following heatshock, there is a marked increase in production of Hsp70 andmuch of this Hsp70 rapidly migrates to the nucleus of the celland concentrates in the nucleoli (Welch and Feramisco, 1984;Pelham, 1984). Upon recovery from heat shock, the Hsp70slowly returns to the cytosol. Although this phenomenon wasdescribed many years ago, its mechanism is only beginning tobe deciphered. Mutations in two regions of Hsp70 reduce itsability to migrate to the nucleus (Knowlton, 1999; Knowlton,2001), whereas phosphorylation of tyrosine 524 increasesmigration (Knowlton et al., 2000). However, little is knownabout the rates at which Hsp70 enters and leaves the nucleusduring the various phases of heat shock. For example, it is notknown whether Hsp70 in the nucleus of stressed or unstressedcells is in rapid equilibrium with Hsp70 in the cytosol; nor isit known whether the interaction of Hsp70 with diffuse proteinsin the nucleus differs from its interaction with proteins that areconcentrated in the nucleolus.A similar question arises in regard to the interaction of Hsp70 with protein aggregates that form in a number of degenerative neurological disorders. A wide array of neurodegenerative diseases, including Alzheimer’s disease,Parkinson disease, prion disease, amyotrophic lateral sclerosis,and Huntington’s disease (HD) show a common feature –inclusion and deposition of abnormal protein in neurons of vulnerable brain regions (Taylor et al., 2002). In HD, theseprotein inclusions are caused by the accumulation of proteinsor protein fragments that contain long stretches of glutaminesthat, in turn, result from a pathological expansion of the CAGrepeat in the coding region of the huntingtin (  Htt  ) gene. In HDand in other ‘polyglutamine’ neurodegenerative diseases,polyglutamine-containing fragments of disease-proteinaccumulate in inclusions found both in the nucleus andcytoplasm. These inclusions might be protective, isolating theaggregated proteins from the rest of the cell (Yang et al., 2002;Kawaguchi et al., 2003; Taylor et al., 2003a). However, theseinclusions might cause harm by sequestering proteasomes, 4991 The molecular chaperone Hsp70 interacts with misfoldedproteins and also accumulates in the nucleus during heatshock. Using GFP-Hsp70 and fluorescence recovery afterphotobleaching, we show that Hsp70 accumulates in thenucleus during heat shock not only because its inflow rateincreases but also because of a marked decrease in itsoutflow rate. Dynamic imaging also shows that GFP-Hsp70has greatly reduced mobility when it interacts withorganelles such as nucleoli in heat-shocked cells or the largeinclusions formed from fragments of mutant huntingtinprotein. In heat-shocked cells, nucleoplasmic Hsp70 hasreduced mobility relative to the cytoplasm, whereas theATPase-deficient mutant of Hsp70, Hsp70(K71E), is almostcompletely immobilized both in the nucleoplasm and thecytoplasm. Moreover, the Hsp70 mutant shows reducedmobility in the presence of diffusive huntingtin fragmentswith expanded polyglutamine repeats. This provides strongevidence that Hsp70 interacts not only with organelles butalso with diffusive proteins in the nucleoplasm andcytoplasm during heat shock as well as with diffusivehuntingtin fragments. Key words: Hsp70, Mobility, Heat shock, Huntingtin Summary Hsp70 dynamicsin vivo: effect of heat shock andprotein aggregation Xian-Chun Zeng 1, *, Samir Bhasin 1, *, Xufeng Wu 1 , Joeng-Goo Lee 1 , Shivani Maffi 1 , Christopher J. Nichols 1 ,Kyung Jin Lee 1 , J. Paul Taylor 2 , Lois E. Greene 1 and Evan Eisenberg 1,‡ 1 Laboratory of Cell Biology, NHLBI, NIH, 50 South Drive MSC 8017, Bethesda, MD 20892-0301, USA 2 Neurogenetics Branch, NINDS, NIH, 10 Center Drive, MSC 1250, Bethesda, MD 20893-1250, USA *These authors contributed equally to this work ‡ Author for correspondence (e-mail: eisenbee@nhlbi.nih.gov) Accepted 21 June 2004 Journal of Cell Science 117, 4991-5000 Published by The Company of Biologists 2004 doi:10.1242/jcs.01373  Research Article  4992 Hsp70, transcription factors or proteins involved in endocytosis(Taylor et al., 2003b; Zhou et al., 2003).When Htt fragments containing expanded glutamine repeatsare expressed in tissue culture cells they form cytoplasmicinclusions that resemble aggresomes (Waelter et al., 2001).Production of aggresomes has been proposed to be ageneralized response of cells to the formation of aggregatedproteins (Johnston et al., 1998; Garcia-Mata et al., 1999).Aggresomes have a number of interesting properties: they aregenerated by transport of small aggregates of misfolded proteintoward the microtubule organizing center (MTOC); in additionto the aggregated protein itself, they recruit a number of different molecular chaperones including Hsp70 and J-domainproteins (homologs of the DnaJ family of proteins) that presentsubstrates to Hsp70; and they are associated with proteasomessuggesting that they may be centers where aggregated proteinsare degraded (Garcia-Mata et al., 1999).There is strong evidence that Hsp70 and J-domain proteinsplay an important role in protecting cells from the deleteriouseffects of the misfolded, aggregated proteins found inassociation with neurodegenerative diseases. For example,overexpression of Hsp70 suppresses degeneration andimproves motor function in a transgenic mouse model of SCA1(Cummings et al., 2001). Similarly, overexpression of Hsp70strongly suppresses the toxicity of expanded polyglutamineand mutant α -synuclein in  Drosophilamelanogaster  models of SCA3 and Parkinson’s disease, respectively (Warrick et al.,1999; Auluck et al., 2002). Hsp70 associates with aggregatedproteins in these diseases in what has been interpreted as aneffort to rid the cell of the offending protein (Muchowski,2002). However, it is not clear whether the protective effect of Hsp70 is because of direct interaction with misfolded proteinor, alternatively, a result of Hsp70’s inhibitory activity onapoptosis (Taylor et al., 2003a; Schaffar et al., 2004).A recent study reported that the mobility of green fluorescentprotein (GFP)-Hsp70 interacting with polylutamine inclusionsor nucleoli (during heat shock) was the same as that of GFP-Hsp70 in the cytosol of untreated cells (Kim et al., 2002).Moreover, this mobility was only about one-fourth of thatfound using truncated Hsp70 lacking a substrate binding site,which, presumably, diffuses freely in the cytosol. This issomewhat surprising because the mobility of freely diffusingproteins is typically orders of magnitude faster than themobility of proteins bound to cellular organelles; in the lattercase the mobility would presumably be a measure of the rateof dissociation of the protein form the organelle (Coscoy et al.,2002). Because Hsp70 associated with the inclusions showedabout the same mobility as Hsp70 interacting with unfoldedproteins in the cytosol, Kim et al. suggested that Htt inclusionsare dynamic rather than static structures, an unexpected findingbecause the Htt in these inclusions, like ataxin3 in itsinclusions, is completely immobilized (Kim et al., 2002; Chaiet al., 2002). However, ataxin 1 inclusions show both fast andslow exchanging components (Stenoien et al., 2002).In the present study, we investigated a number of outstandingquestions involving Hsp70 using fluorescence recovery afterphotobleaching (FRAP) with GFP-Hsp70 and GFP-Hsp70(K71E), an Hsp70 mutant unable to hydrolyze ATP(O’Brien et al., 1996). Our results showed, first, thataccumulation of GFP-Hsp70 in the nucleus during heat shock is caused not only by an increase in the rate of Hsp70 transportinto the nucleus but also by a significant decrease in its rate of transport out of the nucleus. Second, in contrast to the resultsof Kim et al. (Kim et al., 2002), GFP-Hsp70 does not alwaysshow the same mobility nor does it always show the sameinteraction with organelles. The mobility of GFP-Hsp70 boundto either nucleoli during heat shock or polyglutamineinclusions is much less than the mobility of GFP-Hsp70 in thecytosol of control cells, and whereas GFP-Hsp70 is localizedthroughout the nucleoli, it only binds to the surface of theinclusions. Finally, we found that Hsp70 not only interacts withnucleoli during heat shock but also with diffuse cytoplasmicand nucleoplasmic proteins. Similarly it not only interacts withpolyglutamine inclusions, but also with diffuse polyglutaminefragments in the nucleus. This latter interaction might play akey role in the ability of Hsp70 to protect cells from the effectsof heat shock and aggregated Htt fragments. Materials and Methods Plasmids Hsp70 and Hsp70(K71E) (Rajapandi et al., 1998) were sublconedfrom pET21a into pEGFP-C3 (Clontech, CA) or into pFlag-CMV-2(Sigma) using  Hind  III and  Bam HI sites. The Htt polyglutamineconstructs containing glutamine repeat expansions with 25 (Q25) or72 glutamines (Q72) had either an enhanced GFP (EGFP)-tag or amyc-tag on the C-terminal end. The Htt constructs were sublconedinto the N1-RFP vector, obtained as a gift from R. Tsien (StanfordUniversity, Palo Alto, CA) (Campbell et al., 2002). The EGFP-Httconstructs and the EGFP-tagged cAMP response binding protein(CBP) were gifts from A. Tobin (UCLA) and H. Paulson (Universityof Iowa), respectively. Tissue culture and immunostaining HeLa cells were maintained in Dulbecco’s modified Eagle’s medium(DMEM) (Biofluids, MD) supplemented with 10% fetal bovineserum, 2 mM glutamine, penicillin (100 unit/ml) and streptomycin(100 unit/ml) in a humidified incubator with 5% CO 2 at 37°C. Cellswere heat shocked in a humidified incubator with 5% CO 2 at 43°Cfor 1 hour and either imaged immediately after removal from theincubator or returned to the 37°C incubator to follow recovery fromheat shock. Cells were treated with 1 mM or 5 mM dithiothreitol(DTT) following heat shock for 15 minutes at 37°C (Nehls et al.,2000). Cells were transfected with the plasmid DNA using Fugene6(Roche Diagnostics, IN). Cotransfections using a GFP-vector andeither a myc-tagged or flagged-tagged vector were always done usinghalf as much of the GFP-vector as the non-fluorescent vector.Apoptosis was measured 72 hours after transfection by fixing andstaining the cells with cleaved caspase 3 antibody (Cell SignalingTechnology, MA). Confocal microscopy Cells grown on two-chamber 25-mm 2 cover slips (Labtek, NY) wereimaged on a Zeiss LSM 510 confocal microscope. GFP-clathrin wasimaged and photobleached using 488-nm laser light with a 40  , 1.4NA objective. RFP-Htt was imaged using a 543-nm laser light. Adefined region was photobleached at high laser power resulting in 50-80% reduction in the fluorescence intensity. The fluorescencerecovery was monitored by scanning at low laser power. To get timepoints in the millisecond time range when measuring the fluorescencerecovery of photobleached, highly diffusable protein, the fluorescencescanning was modified in two major ways which decreased the scantime. First, the pixel density was decreased from 512  512 to128  128. Second, the scanning distance along the ordinate axis was Journal of Cell Science 117 (21)  4993Hsp70 dynamics in vivo markedly reduced. When data sets were compared, identicalconditions were used in photobleaching the cells including the numberof bleaches, the area of the photobleach region and the time course of imaging at low laser power. Data analysis For each experimental condition, a minimum of ten data sets wereaveraged to get the mean and standard deviation for each time point.The fluorescence-intensity data in each experiment were normalizedby setting the maximum fluorescence to 100% and the minimum to0%. The very low laser intensity used in scanning the cell after theinitial photobleach did not cause significant bleaching during ourexperiments and therefore no correction was necessary for this effect.Even though the fluorescence of the total GFP pool in the cell wasunaffected by scanning, the total recovery in most experiments wasonly about 80% of the initial fluorescence because about 20% of thetotal GFP pool in the cell was bleached by the initial bleach. Results Characterization of GFP-Hsp70 To study the properties of Hsp70, we constructed an N-terminalGFP-Hsp70 and then established whether it still maintainsnormal Hsp70 activity. One of the activities of Hsp70 as amolecular chaperone is to protect the cell against the toxiceffects of Htt-aggregation (Merienne et al., 2003). Therefore,we examined whether GFP-Hsp70 is able to protect the cellagainst the apoptotic effects of polyglutamine aggregation. Wefound that GFP-Hsp70 protected cells to a similar extent asflagged-tagged Hsp70 from apoptosis induced by expression of Htt(Q72) (Fig. 1A). As expected, expression of Htt(Q25) didnot cause significant apoptosis of transfected cells. This clearlydemonstrates that GFP-Hsp70 is physiologically active. Another way of validating that GFP-Hsp70 retains theactivity of unmodified Hsp70 is to show that it migrates to thenucleus and, in particular, to nucleoli following heat shock.Fig. 1B shows the distribution of GFP-Hsp70 expressed inHeLa cells before and after heat shock. Before heat shock about 60% of the GFP-Hsp70 was present in the cytosol,although some was also present in the nucleus, particularly incells showing a high overall expression of GFP-Hsp70.However, even in these cells, the GFP-Hsp70 was completelyexcluded from the nucleoli before heat shock. By contrast,immediately following heat shock, there was a marked increasein the level of GFP-Hsp70 present in the nucleus andfurthermore, much of this GFP-Hsp70 became concentrated inthe nucleoli.It is not clear whether the changes in nuclear concentrationsof Hsp70 during heat shock are only owing to changes in theinflow rate of Hsp70 or whether both inflow and outflow rateschange during heat shock and recovery. Because GFP-Hsp70can be photobleached, we addressed this question bymeasuring the rates of Hsp70 entry into and exit from thenucleus under different conditions. Completely bleaching thetotal volume of either the cytosol or the nucleus (see Fig. 2legend) allowed us to determine the percent of Hsp70 thatflowed into or out of the nucleus over a given period of time.Before heat shock, Hsp70 clearly passed across the nuclearmembrane in both directions, its rate of transport was relativelyslow (Fig. 2A-C). Quantification of these data by averagingover a large number of cells showed that in 15 minutes about6% of the Hsp70 in the cytosol passed into the nucleus andabout 40% of the Hsp70 in the nucleus passed into the cytosol(Fig. 3). Given that the cytosol contains about 60% of the totalHsp70 in the cell and assuming that the nucleus is about one-sixth of the total cell volume, this means that roughly equalamounts of Hsp70 are passing in both directions across thenuclear membrane, as expected under steady-state conditions.Fig. 2D-F shows a similar experiment carried outimmediately after heat shock. Quantification of these data inFig. 3 shows that following heat shock the inflow rate of Hsp70into the nucleus doubled, while the outflow rate was reducedby about two-thirds. Thus, the rapid net accumulation of Hsp70in the nucleus following heat shock results not only fromincreased inflow, but also from markedly reduced outflow. Thisresults in the nucleus being much brighter than the cytosol (Fig.4A). Six hours following heat shock, during the recoveryphase, the outflow rate increased beyond the basal level inuntreated cells while the inflow rate decreased markedly. Bycontrast, the transport of GFP across the nuclear membrane,which is very rapid, was unaffected by heat shock (data notshown). It should be noticed that comparison of inflow ratesand outflow rates necessitates a correction for the relativevolume of the nucleus and cytosol. However, comparison of the relative values of inflow and outflow rates requires only that Fig. 1. Properties of GFP-Hsp70. (A) Protection of GFP-Hsp70against apoptosis from the expression of Htt(Q72). The extent of apoptosis was measured 72 hours after transfection in control cellsand cells expressing GFP-Htt(Q25), GFP-Htt(Q72), GFP-Htt(Q72)and flag-tagged Hsp70, myc-tagged Htt(Q72) and GFP vector, andmyc-tagged Htt(Q72) and GFP-Hsp70. Apoptosis was measured byimmunostaining for cleaved caspase 3 using rhodamine-conjugatedsecondary antibody. The data represent an average of threeexperiments in which 100 GFP cells were analyzed for positivecaspase staining. (B) Distribution of GFP-Hsp70 in control (left) andheat-shocked cells (right).  4994 the relative volumes of the nucleus and cytosol remain constantfollowing heat shock. Therefore, these data show that the levelof Hsp70 in the nucleus, under various conditions, is controlledby changes in both inflow and outflow rates of Hsp70 from thenucleus.In contrast to the results obtained with GFP-Hsp70, themigration of the ATPase-inactive mutant of Hsp70, GFP-Hsp70(K71E), was much less affected by heat shock. Beforeheat shock, the distribution of GFP-Hsp70(K71E) was verysimilar to GFP-Hsp70 (Fig. 2G-I). However, after heat shock there was no significant accumulation of GFP-Hsp70(K71E) inthe nucleus and nucleolus (Fig. 2J-L), indicating that ATPbinding or hydrolysis must play an important role in Hsp70migration. Quantification of the in- and out-rates shows thatGFP-Hsp70(K71E) entered into and exited from the nucleusmuch more slowly than GFP-Hsp70 in non heat-shocked cells(Fig. 3). Furthermore, following heat shock, the rate of GFP-Hsp70(K71E) entry into and exit from the nucleus becameunmeasurably slow, so that there was almost no change in thedistribution of GFP-Hsp70(K71E) between the nucleus and thecytosol following heat shock (Fig. 4B). Thus, particularly inheat shock, the ability of Hsp70 to rapidly flow into and outofthe nucleus depends on an intact ATP-hydrolysis-site.Furthermore, we found that expression of flag-tagged Hsp70(K71E) in control cellsmarkedly decreased the rates at which GFP-Hsp70(K71E) itself entered and exited fromthe nucleus (data not shown). This dominantinfluence suggests that Hsp70(K71E) mightform a complex with GFP-Hsp70(K71E) andprevent it from being transported into and outof the nucleus in a normal manner. Mobility of Hsp70 during heat shock We next investigated the local mobility of GFP-Hsp70(K71E) in control and heat-shocked cells by determining its rateofrecovery-of-fluorescence followingphotobleaching. We first determined themobility of GFP alone, which is consideredcompletely mobile. We obtained a half-life of recovery of 0.3 seconds for fluorescencerecovery of GFP in the cytosol (Fig. 5A), butbecause photobleaching of the freelydiffusable material did not just occur in thevolume that we bleached, the fluorescencerecovery is not just a single exponential(Coscoy et al., 2002). Therefore, themeasured half-life does not provide an accurate diffusionconstant. Instead, to compare different recovery rates, we usedthe measured half-lives of fluorescence recovery as a relativemeasure, comparing all of the other half-lives for fluorescencerecovery to the half-life for GFP alone, using identical settingswhen performing the photobleaches. We first measured themobilities of Hsp70 in the cytoplasm and nucleus and foundthat in all cases the half-life of fluorescence recovery was lessthan 1 second. The mobilities of GFP-Hsp70 in both thecytoplasm (Fig. 5A) and nucleoplasm (Fig. 5B) were less thana factor of 2 higher than the mobility of GFP. This slightlyhigher value can in part be attributed to the three-fold highermolecular weight of GFP-Hsp70 compared to GFP becausediffusion is roughly proportional to the cube root of themolecular weight (Reits and Neefjes, 2001). In the nucleus,theplateau level of GFP-fluorescence-recovery afterphotobleaching is slightly lower than the plateau level of GFP-Hsp70-fluorescence-recovery because, as described in Materialand Methods, as we bleach, fluorescent material outside of thebleach flows into the bleached area, and because GFP has ahigher mobility than GFP-Hsp70, this effect occurs to a greaterextent with GFP than with GFP-Hsp70.We next investigated the mobility of GFP-Hsp70 after heat Journal of Cell Science 117 (21) Fig. 2. The transport of (A-F) GFP-Hsp70 and (G-L) GFP-Hsp70(K71E) into and out of the nucleusin control (A-C,G-I) and heat-shocked cells(D-F,J-L). Cells before (A.D,G,J) photobleaching,immediately after (B,E,H,K) photobleaching and15 minutes following (C,F,I,L) photobleaching.Outlined areas indicate photobleached regions.Fluorescence of pre-bleached cells was intensifiedduring imaging to insure that the fluorescencesignal of the post-photobleached cells wassufficiently high to accurately measure transport.

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Jul 23, 2017
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